Achyrocline satureioides (Lam.) DC.

The commercial exploitation of this plant is carried out through the recollection from the wild. INIA in Montevideo, Uruguay also cultivates it.

Crude, semi-processed

Argentinean and Brazilian Pharmacopeias

The cultivation should be based on the use of seeds recollected from old plants in order to have higher germination (Marquez and Barrios, 1997). The propagation through stakes is recommended using apical segments of the branches taken from the mother plant in the period of vegetative growth (Davies, 1992).

The cultivation normally allows two harvest: the first, after one year of transplantation to the field (March and April-Uruguay) and the second, the following year in the same period. Harvesting is done manually cutting inflorescences and leaves (Melillo de Magalhaes, 2000). Drying is carried out by hanging the plants in tubes in dryers with air circulation at 40° C or under shade.

In Argentina, infusion of leaves and branches with flowers to treat digestive problems. Antiasthmatic, tonic, stimulant febrifuge and antihelmintic. In Paraguay, aerial parts are used for infections. In Colombia, is used for tumors whereas in Venezuela, whole plant is used to treat diabetes, impotence and emenagogue (Gupta, 1995).

In Uruguay, infusion of inflorescence and floral stems are used as colagogus, carminative, anti-inflammatory, antiflogistic, emenagogue, sedative, hipocholesterolemic. It is also used externally as antiseptic and anti-inflammatory (Gupta, 1995). It is also used as expectorant, sudorific, febrifuge (Correa and Bernal, 1990). Decoction is used for skin bath. It is used for acne, epidermic diseases and malign tumors (García-Barriga, 1975; Rodriguez, 1983). Bronchial asthma, diabetes, stomach upset (colics, fermentations and heavy sleepy), menstruation regulator, vaginal infections (Manfred, 1977).

Kallawaya use Leaves, flowers, dried and fresh stems in infusion for cough. Decoctions against lung problems and decoction against colics (Girault, 1987). The inflorescences, leaves and dried stems are used internally as digestive infusion, antispasmodic, emenagogue, antiarteriosclerosis, sedative, antiseptic, diuretic and anti-inflammatory (Alonso, 2004).

In Bolivia, Peru and Argentina, this is used as antiasmatic and antitusive. Externally, it is used for vaginal bath. In Brazil, it is used as antidiarreic, hypoglycemic, antidisenteric. As sirup is recommended for cold and flu. In Colombia, infusion of whole plant is used externally in case of tumors.

Anti-inflammatory and antispasmodic activities have been demonstrated (Langeloh et al., 1981-1982; Simões et al., 1988a; b). Also contains polysaccharides with immune-stimulating action (Wagner et al., 1985; Puhlmann et al., 1992). Anti-herpetic anti-inflammatory, antispasmodic and antimicrobial activities (García et al., 1990; Gutkind et al., 1981; Simoes et al., 1988). The fraction of polysaccharide administered by IP in mice showed inmunostimulant activity and increase the fagocitic activity (Gupta, 1995). Moreover showed genotoxic activity, molluscicidal against Biomphalaria glabrata (100 ppm), analgesic activity, anti-inflammatory activity and antimutagenic activity. Additionally, it showed musle relaxant activity (De Souza et al., 1984).

Antimicrobial activity 
Antibacterial activity is significant against pathogenic flora, which affects the skin (particularly, Staphylococcus aureus) being responsible of this activity cafeic acid and quercetine present in the aqueous extracts (Schmeda, 1982; Lemos and Oliveira, 1997). The decoction of this plant showed activity against Staphylococcus aureus (MIC 6.25 µg/ml), which is similar to the activities of quinolones (Ciprofloxacine 5 µg/ml, enrofloxacine 5 µg/ml, balofloxacine 5 µg/ml). Another microorganismo susceptible is Micrococcus luteus. This extract was not active against Bacillus subtilis and Pseudomas aeroginosa and pathogenic fungus (Gutkind et al., 1981; Pérez and Anesini, 1994).

The 95% ethanolic extract and hexane-ethyl acetate extract of whole plant showed in vitro molluscicidal activity against Biomphalaria glabra at 100 ppm (De Souza et al., 2001). The 80% extract of aerial parts did not present inhibitory activity of Herpes simplex 1 infected cells (García et al., 1990). In contrast, the hydroalcoholic extract from flowers displayed antiviral activity against Herpes simplex 1 y 2, polivirus, rhinovirus, type 14, HIV, Vesicular stomatitis virus. In this case, poliphenolic acids and triterpenic saponines were responsible for the activity. In this regard, saponines of olean group inhibited the synthesis of HSV-1 viral DNA (Van den Berghe et al., 1985; Navarrete, 1989; Simoes, 1994).

The aqueous extract of flowers (conc. 50 mg/ml) presented in vitro antiretroviral against HIV. In this regard, 1-MO-3, 5-dicaffeoylquinic acid (DCQA) have been identified as responsible for the activity. This compound in concentrations from 0.06-0.66 µg/ml inhibited integrase, which is coupled to viral replication system (Robinson et al., 1996). In the related specie A. alata , the principal antiherpetic compound was 3-methoxyflavone with inhibitory activity of 99.8%, whereas, from A. flaccida, three compounds: 3-methylether quercetine, 4,2,4-trihydroxy-6-methoxy chalcone and 7,4-di-hydroxy-5methoxy flavone (García et al., 1990). The ethylacetate extract of marcela did not present antimalarial activity against Plasmodium berghei when mice were infected at intragastric dosis of 100 mg/kg (Brandao et al., 1985).

The ethanolic extract of flowers and leaves of this plant at concentration of 250 mg/ml showed in vitro litic effect against Trypanosoma cruzi. The hexane extract of flowers showed insecticidal activity (45% of mortality) against Triatoma infestans, vector of Trypanosoma cruzi (Rojas de Arias et al., 1995).

Analgesic and anti-inflammatory activity 
95% ethanolic and aqueous extracts showed local anti-inflammatory activity at dosis of 75 mg/ear, which is a joint action of flavonoids–methyl ether quercetine, luteoline and quercetine (Simoes, 1988a; Wagner and Ott, 1991; Handa, 1992). Alcoholic and aqueous extracts administered IP at doses of 200 mg/Kg and 75 mg/kg, respectively showed activity (Simoes, 1988a). 95% ethanolic extract and aqueous of inflorescence at doses of 200 mg/kg, IP and 75 mg/kg, IP, respectively showed analgesic activity (Simoes, 1988a). Methanol, water, ethyl acetate and benzene extract did not show inhibitory effect on 5-lipooxigenase related with inflammation via and leucotriene formation (Matsunaga et al., 2000).

The fraction of polysaccharides administered IP in mice at dosis of 10 mg/kg exhibited inmunestimulant activity demonstrated through an increment in the fagocitic activity of PMN leucocytes (Wagner et al., 1985). The aqueous and 95% ethanolic extracts of flowers demonstrated antispasmodic in the yeyune isolated from rats under contractile induction with acetic and barium acids (De Souza et al., 1984; Filot da Silva and Langeloh, 1994) and sedative properties due to antibarbituric activity (Simoes et al., 1986). The ethanolic and aqueous extracts of flowers were administered via intragastric to rats at dosis of 200 mg/kg, did not show intestinal motility (Simoes, 1988b). The aqueous extract of Marcela demonstrated the in vitro inhibition of glycan compounds (metilglioxal) involved in diabetic complication (Gugiucci and Menini, 2002a). The cafeic acids and derivatives are responsible for effect colagogues and coleretic (Broussalis et al., 1996).

Intragastric administration of hydroalcoholic extract of this plant at dosis of 400 mg/kg did not show any activity (Broussalis et al., 1996). Potasium depletion has not been observed in the urinary excretion of rats after administration of 400 mg/kg of hydroalcoholic extract of this plant (Rocha et al., 1994). At cardiovascular level, the aqueous extract (10% P/V lyophilized) produced arterial hypotension (concentration dependent) in rats with anesthesia rats (Vecchio et al., 2002).

The infusion of aerial parts at concentrations of 0.1 mg/ml showed antioxidant and hepatoprotective in rats due to inhibition of lipid peroxidation and coleretic effect (Desmarchelier et al., 1997; Kadanian et al., 2001). Equally, at concentration of 5 µg/ml of aqueous extract (equivalent to dilution of 1/100 of a normal infusion) showed antioxidant in the oxidative tests under cooper, peroxinitrite and oxidation of LDL (Gugliucci and Menini, 2002b). The antioxidant activity of flavonoid fraction (luteiline, quercetine and 3-O-methylquercetine), proved also in infusions showed cytoprotective effect in neuronal cultures. The range of cytoprotection for luteoline was 50-250 µM and quercetine 50-50 µM (Arredondo et al., 2003).

Plant contains essential oil with α and β-pinene (Bauer et al., 1979), flavonoids and other phenolics (Hänsel et al., 1971; Ferraro et al., 1981; Broussalis et al., 1988) and one kawa type pyrone (Kaloga et al., 1983).

The essential oil from this plant contains 1,8-cineol, caryophyllene, caryophyllene oxide, isognaphaline (Hänsel and Ohlendorf, 1971), protocatequilcalearinine, caffeoylcalerianine, δ-cadinene, caffeic acid, cariatine, italidipyrone, germacrene-D, quercetin, quercetagetin, lauricepyrone, α-pinene, tamarixetine, alnustine (Gupta, 1995). In the aerial parts, sesquiterpene, phenylpyrone derivatives, germacrene-D, 3,7-dimethoxy-5-8-dihidroxyflavone, 23-methyl-6-O-desmethylauricepyrone and italidipyrone, morin; in flowers: flavonoids, polyphenolic acids and kawapyrone type of compound; in the roots: acetylenic compounds. Other compounds found in this plant are galangin, galangin-3-methylether, quercetin, quercetin-3-methyl-ether, luteolin, caffeic acid, protocatechuic acid and two esters of calleryanin (3,4-dihidroxy-bencilalcohol-4-glycoside) (Ferraro et al., 1981) 6-4’-hidroxy-trans-styryl)-4-methoxy-2-pyrone (Kaloga et al., 1983).

In addition compounds found in this plant are 23-methyl-6-O-desmethyllauricepyrona-6, italidipyrone-7 (Schmeda, 1984), alnustine, 7-hydroxi-3,5,8-trimethoxyflavone, 3,5,7,8-tetramethoxyflavone and 5,7,8-trimethoxyflavone (Mesquita et al., 1986) are reported in this plants.

Microscopic descriptions of A. satureioides (powder) has been published in the Brazilian Pharmacopoeia, 2002, which also gives the following additional qualitative and quantitative parameters.

Identification by thin layer chromatography 
Apply separately, 5-10 µl of sample solution and 2-3 µl of reference solution on cellulose chromatographic plates, which are developed in chloroform-acid acetic glacial-water (50:45:5) as mobile phase.

Sample solution 
Reflux 10 g of the drug in 100 ml of distilled water for one hour. Filter. Use a separatory funnel to extract and the filtrate is partitioned with ethyl acetate (4 x 25 ml). Wash the ethyl acetate fraction with water and dry with anhydrous sodium sulphate. Evaporate in a rotavapor. Dilute the residue in 15 ml of methanol and proceed with chromatographic analysis.

Reference solution
Dissolve 1 mg of each one of the standards (quercetin, 3-O-methyl-quercetin, luteolin and caffeic acid) separately in 100 μl of methanol. Run the chromatoplate up to 10 cm approximately. Dry at room temperature. Examine the chromatoplate under the ultraviolet light (354 nm). Spray with ethanolic solution 1% (p/V) of amino-ethanol diphenylborate (Natural Products Reagent). Additionally spray with ethanolic solution 5% (p/V) of polyethylenglycol 400. The chromatogram shows a yellow dark fluorescent spot the same distance as the reference solution (Rf 0.40) corresponding to quercetin, another at Rf 0.60 (luteolin) of brown color, a spot at Rf 0.80 of clear brown color attributed to 3-O-methyl-quercetin, and another spot of fluorescent blue (Rf 0.90) corresponding to caffeic acid.

Purity assays 

• Foreign matter: not more than 2%
• Determination of water: not more than 10%
• Total ashes: not more than 8%

Quantifitation 
Proceed as described in the Determination of Essential oils (V.4.2.6.) Use a flask of 2000 ml with sufficient water to cover the plant drug. Use 30 g of flowers and distill for 5 h. After distillation, immediately note the volume of essential oil.

Determination of total flavonoids
Weigh exactly 0.400 g of pulverized drug (800 µm) and place in a flask of 100 ml. Add 1 ml of solution of hexamethylenetetramine 0.5% (w/v), 20 ml of acetone and 2 ml of hydrochloric acid. Heat in a water bath, with a reflux system, for 30 min. Filter the mixture and pass it to a volumetric flask of 100 ml. Wash the residue of the drug and place it in a flask, with two portions of 20 ml of acetone, reflux for 10 min. Then chill to room temperature, filter the solution and pass it to a volumetric flask, to complete its volume with acetone. In a separatory funnel, place 20 ml of the solution with 20 ml of water and then extract with 15 ml of ethyl acetate, repeating three times with 10 ml of ethyl acetate. Mix the phases of ethyl acetate and wash them in a separation funnel with two portions of 50 ml of water, then transfer to a volumetric flask of 50 ml, make up the volume with ethyl acetate (solution SM). Pippet 10 ml of this solution, add 1 ml of aluminum chloride reagent, dilute in a volumetric flask of 25 ml with a methanolic solution of acetic acid 5% (v/v). Prepare a blank by diluting 10 ml of SM to bring up to 25 ml with methanolic acetic acid 5% (v/v). After 30 min, measure the absorbance of the solution at 425 nm in a cell of 1 cm, using the blank to calibrate zero. Calculate the total flavonoid percentage according to the following formula:

Where A = absorbance 
m = weight of drug (g); 
Pd = loss on drying (%; p/p)

The result is expresed in percentage (p/p) of total flavonoids calculated as quercetin (C15H10O7).

Determination of quercetin and luteolin 
Proceed according to the procedure described for high-pressure liquid chromatography (V.2.17.4). Extract 18 g of powdered dry drug (800 µm) in Soxhlet with 300 ml of n-hexane for 3 hours. The defatted material is extracted with 300 ml of ethyl acetate for 3 hours. Ethyl acetate extract is concentrated to dryness in a rotavapor and the residue is dissolved quantitatively in methanol. The solution is transferred to a volumetric flask of 10 ml and brought to the volume with methanol. An aliquot of 1 ml of this solution is diluted to 50 ml with the same solvent. An aliquot of 4 ml of this solution is diluted to 20 ml, using as solvent a mixture of methanol:water (53:47). The samples are filtered through a filter of polyvinylidene fluorate membrane (0.45 µm) and injected into a chromatograph. The extracts are injected in triplicate and the results are expressed in grams of quercetin and luteolin per 100 grams of drug (%, w/w).

Calibration curve 
Five mg each of the reference substances, quercetin and luteolin, are exactly weighed and dissolved in methanol. The solution is transfered to a volumetric flask of 100 ml with methanol. Aliquots of the mother solution are diluted with a mixture of methanol:water (53:47), obtaining solutions with content of quercetin and luteolin, in the following concentrations: 1.5; 2.5; 5; 7.5; 10 µg/ml. The solutions are filtered through a filter of polivinylidene fluorate membrane (0.45 µm) and injected into the chromatograph.

Chromatographic conditions: The chromatographic analysis is carried out in a chromatograph with UV detector. The chromatographic conditions are: octadecylsilizade silica gel pre-column of 10 µm; column of stainless steel (250 mm x 4 mm i.d.) packed with octadecylsilizade silica gel of 5 µm. Mobile phase: methanol: solution of phosphoric acid 1% (w/v) in a proportion of 53:47; flow of 0.6 ml/min; detector at 362 nm. Mobile phase previously filtered through polyvinylidene fluorate membrane 0.45 µm of pore diameter. Determine the area of the peak of quercetin and luteolin using a calibration curve.

Preservation and storage: Store in well covered containers, protected from light and heat for a period not more than a year.

At recomended doses, no toxic effects are observed. At dosis of 500 mg/kg of IV administration of 95% ethanolic extract and aqueous extract, no toxic effects were observed (Simoes et al., 1988). No negative effects on rats reproduction have been reported (Krein et al., 1996).

Aqueous extract exhibited genotoxicity with Escherichia coli PQ37 (conc. 10 mg/plaque) and mutagenic for Salmonella typhimurium TA-98 (conc. 50 mg/plaque), TA-100 (conc. 200 mg/plaque), TA-102 (conc. 50 mg/plaque) and Aspergillus nidulans (Vargas et al., 1990; Gonzalez et al., 1993; Ferrari et al., 1993). Hot aqueous extract of inflorescences and dried stems of this plant were active in the Ames test, in 1% concentration. This extract inhibited the growth of the roots of Triticum aestivum at 5%. In minor concentrations (0.5%), the results were considered doubtful (Gonzalez et al., 1993).

The aqueous extract administered intraperitoneally in mouse at doses of 200 mg/kg, potentiated the activity of barbiturates (Simoes et al., 1988).

• Infusion: 10 g/l from flowers. Administered 2-3 cups daily.
• Tincture: 20 g of inflorescence in 100 ml of ethanol 60%. 30-40 drops, 2-3 times per day.
• External use: Infusion of 30 g of flowers in 1 liter of water. Applied as a compress, 1-3 times per day.
• Phytocosmetic: Glycolic extract (2-5%) is used to prepare shampoos and soaps, 1-3 times per day. 5% infusion is used to lighten hair color (Alonso, 2004).

This plant is found in the Argentine Food Code since 1995 (used in bitter aperitif) and recognized in the list of approved herbs for Human use (Resol. 2673/99). Moreover, it is inscriptive in the Brazilian Pharmacopea (Fascicule III, 2001) and included in the list of species of Free sale in the herbalists of Uruguay, under ordinance No. 445.